Ezrin增强子敲除可抑制人食管癌Eca-109细胞的增殖和迁移
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摘要
目的:探讨ezrin增强子敲除对食管癌Eca-109细胞ezrin基因表达、细胞增殖和迁移的影响。方法:将靶向ezrin增强子上、下游的CRISPR/Cas9重组质粒共转染食管癌Eca-109细胞,经嘌呤霉素筛选,获得敲除ezrin增强子的细胞株Eca-C2。用qPCR和Western blotting分别检测敲除ezrin增强子的Eca-C2细胞中ezrin mRNA和蛋白的表达,用蛋白芯片技术检测MAPK通路相关蛋白的表达,用WST-1法和细胞划痕愈合实验分别检测ezrin增强子敲除对Eca-C2细胞增殖和迁移能力的影响。结果:成功构建稳定敲除ezrin增强子的食管癌细胞株Eca-C2;与对照细胞相比,ezrin增强子敲除细胞ezrin mRNA和蛋白的表达水平均明显降低(均P<0.05)。Eca-C2细胞中17种被检测的MAPK通路相关蛋白中有9种(AKT、CREB、GSK3b、MKK6、m TOR、P38、P53、P70S6K和RSK1)表达下调,ezrin增强子敲除后细胞的增殖和迁移能力受到明显抑制(均P<0.05)。结论:在人食管癌Eca-109细胞中,敲除ezrin增强子可明显抑制细胞的增殖和迁移。
Objective: To investigate the effects of ezrin enhancer knockout on ezrin gene expression, cell proliferation and migration of human esophageal carcinoma Eca-109 cells. Methods: The CRISPR/Cas9 recombinant plasmids targeting upstream/downstream of human ezrin enhancer were co-transfected into human esophageal carcinoma Eca-109 cells, and the cell line Eca-C2 with ezrin enhancer knockout was screened by purinomycin. Then the expression levels of ezrin mRNA and protein in Eca-C2 cells were detected by Real-time quantitative PCR(qPCR) and Western blotting, respectively; The expression levels of MAPK-pathway-related proteins were detected by protein array technology; and the effects of ezrin enhancer knockout on the proliferation and migration of Eca-C2 cells were analyzed by WST-1 method and wound-healing assay, respectively. Results: The human esophageal carcinoma cell line Eca-C2 with stable ezrin enhancer knockout was established successfully. Compared with control cells, the mRNA and protein expressions of ezrin in Eca-C2 cells were significantly reduced(all P<0.05). Among the 17 detected MAPK pathway related proteins in Eca-C2 cells, 9 proteins(AKT, CREB, GSK3 b, MKK6, m TOR, P38, P53, P70 S6 K and RSK1) were down-regulated, and the cell proliferation and migration were significantly inhibited(all P<0.05). Conclusion: ezrin enhancer knockout can significantly inhibit the cell proliferation and migration of human esophageal carcinoma Eca-109 cells.
Objective: To investigate the effects of ezrin enhancer knockout on ezrin gene expression, cell proliferation and migration of human esophageal carcinoma Eca-109 cells. Methods: The CRISPR/Cas9 recombinant plasmids targeting upstream/downstream of human ezrin enhancer were co-transfected into human esophageal carcinoma Eca-109 cells, and the cell line Eca-C2 with ezrin enhancer knockout was screened by purinomycin. Then the expression levels of ezrin mRNA and protein in Eca-C2 cells were detected by Real-time quantitative PCR(qPCR) and Western blotting, respectively; The expression levels of MAPK-pathway-related proteins were detected by protein array technology; and the effects of ezrin enhancer knockout on the proliferation and migration of Eca-C2 cells were analyzed by WST-1 method and wound-healing assay, respectively. Results: The human esophageal carcinoma cell line Eca-C2 with stable ezrin enhancer knockout was established successfully. Compared with control cells, the mRNA and protein expressions of ezrin in Eca-C2 cells were significantly reduced(all P<0.05). Among the 17 detected MAPK pathway related proteins in Eca-C2 cells, 9 proteins(AKT, CREB, GSK3 b, MKK6, m TOR, P38, P53, P70 S6 K and RSK1) were down-regulated, and the cell proliferation and migration were significantly inhibited(all P<0.05). Conclusion: ezrin enhancer knockout can significantly inhibit the cell proliferation and migration of human esophageal carcinoma Eca-109 cells.
引文
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[19]LI S,WANG Z,HUANG J,et al.Clinicopathological and prognostic significance of mTOR and phosphorylated mTOR expression in patients with esophageal squamous cell carcinoma:a systematic review and meta-analysis[J].BMC Cancer,2016,16(11):877-893.DOI:10.1186/s12885-016-2940-7.
[20]DING Y,LIU W,WANG X,et al.Bufalin induces apoptosis in human esophageal carcinoma ECA109 cells by inhibiting the activation of the mTOR/p70S6K pathway[J].Oncol Lett,2018,15(6):9339-9346.DOI:10.3892/ol.2018.8526
[21]GAO S,LI S,DUAN X,et al.Inhibition of glycogen synthase kinase 3 beta(GSK3[beta])suppresses the progression of esophageal squamous cell carcinoma by modifying STAT3 activity[J].Mol Carcinogenesis,2017,56(10):2301-2316.DOI:10.1002/mc.22685.
[2]ALONSO-LEBRERO J L,SERRADOR J M,DOMINGUEZ-JI-MENEZ C,et al.Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule3 with moesin and ezrin in myeloid cells[J/OL].Blood,2000,95(7):2413-2419[2018-07-08].http://www.bloodjournal.org/content/95/7/2413.long?sso-checked=true.
[3]YOUN J Y,WANG T,CAI H,et al.An ezrin/Calpain/PI3K/AMPK/eNOSs 1179 signaling cascade mediating VEGF-dependent endothelial nitric oxide production[J].Circ Res,2009,104(1):50-59.DOI:10.1161/CIRCRESAHA.108.178467.
[4]XIE J J,XU L Y,WU Z Y,et al.Prognostic implication of ezrin expression in esophageal squamous cell carcinoma[J].J Surg Oncol,2011,104(5):538-543.DOI:10.1002/jso.21909.
[5]LIANG F,WANG Y,SHI L,et al.Association of ezrin expression with the progression and prognosis of gastrointestinal cancer:a meta-analysis[J/OL].Oncotarget,2017,8(54):93186-93195[2018-07-08].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696254/.DOI:10.18632/oncotarget.21473.
[6]SLIK K,KURKI S,KORPELA T,et al.Ezrin expression combined with MSI status in prognostication of stageⅡcolorectal cancer[J/OL].PLoS One,2017,12(9):e0185436[2018-07-08].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617236/.DOI:10.1371/journal.pone.0185436.
[7]GAO S Y,LI E M,CUI L,et al.Sp1 and AP-1 regulate expression of the human gene VIL2 in esophageal carcinoma cells[J].J Biol Chem,2009,284(12):7995-8004.DOI:10.1074/jbc.M809734200.
[8]GAO S Y,DAI Y P,YIN M J,et al.Potential transcriptional regulatory regions exist upstream of the human ezrin gene promoter in esophageal carcinoma cells[J].Acta Biochim Biophys Sin,2011,43(6):455-464.DOI:10.1093/abbs/gmr033
[9]张青峰,卫金岐,张芳婷,等.几种肿瘤细胞中ezrin基因增强子区转录调控特性的研究[J].中国细胞生物学学报,2014,36(5):610-616.DOI:10.11844/cjcb.2014.05.0372.
[10]LAU V,DAVIE J R.The discovery and development of the CRIS-PR system in applications in genome manipulation[J].Biochem Cell Biol,2017,95(2):203-210.DOI:10.1139/bcb-2016-0159.
[11]郭晓龙,张青峰,野庆松,等.靶向ezrin增强子关键区的CRISPR/Cas9载体的构建[J].生物学杂志,2017,34(6):19-22.DOI:10.3969/j.issn.2095-1736.2017.06.019.
[12]ZHONG Z Q,SONG M M,HE Y,et al.Knockdown of ezrin by RNA interference reverses malignant behavior of human pancreatic cancer cells in vitro[J].Asian Pac J Cancer Prev,2012,13(8):3781-3789.
[13]CHYLINSKI K,MAKAROVA K S,CHARPENTIER E,et al.Classification and evolution of typeⅡCRISPR-Cas systems[J].Nucleic Acids Res,2014,42(10):6091-6105.DOI:10.1093/nar/gku241.
[14]XIE J J,XU L Y,XIE Y M,et al.Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells[J].Int J Cancer,2009,124(11):2549-2558.DOI:10.1002/ijc.24216.
[15]谢一方,王永明.基因编辑技术的原理及其在癌症研究中的应用[J].中国肿瘤生物治疗杂志,2017,24(8):815-827.DOI:10.3872/j.issn.1007-385X.2017.08.001.
[16]ZHAI J,WANG Y,YANG F,et al.DRP-1,ezrin and E-cadherin expression and the association with esophageal squamous cell carcinoma[J].Oncol Lett,2014,8(1):133-138.DOI:10.3892/ol.2014.2114.
[17]ZIPPERER A,KRETSCHMER D.Cytotoxicity assays as predictors of the safety and efficacy of antimicrobial agents[J/OL].Methods Mol Biol,2017,1520:107-118[2018-07-08].https://link.springer.com/protocol/10.1007%2F978-1-4939-6634-9_6.DOI:10.1007/978-1-4939-6634-9_6.
[18]CHEN P,LI M,HAO Q I,et al.Targeting the overexpressed CREBinhibits esophageal squamous cell carcinoma cell growth[J].Oncol Rep,2018,39(3):1369-1377.DOI:10.3892/or.2017.6167.
[19]LI S,WANG Z,HUANG J,et al.Clinicopathological and prognostic significance of mTOR and phosphorylated mTOR expression in patients with esophageal squamous cell carcinoma:a systematic review and meta-analysis[J].BMC Cancer,2016,16(11):877-893.DOI:10.1186/s12885-016-2940-7.
[20]DING Y,LIU W,WANG X,et al.Bufalin induces apoptosis in human esophageal carcinoma ECA109 cells by inhibiting the activation of the mTOR/p70S6K pathway[J].Oncol Lett,2018,15(6):9339-9346.DOI:10.3892/ol.2018.8526
[21]GAO S,LI S,DUAN X,et al.Inhibition of glycogen synthase kinase 3 beta(GSK3[beta])suppresses the progression of esophageal squamous cell carcinoma by modifying STAT3 activity[J].Mol Carcinogenesis,2017,56(10):2301-2316.DOI:10.1002/mc.22685.